Escherichia coli, Pseudomonas aeruginosa, Staphylococcus aureus, and Bacillus cereus. In the examined microorganisms, the antibacterial properties of the novel alkyl derivatives increased substantially higher than chitosan. The minimum inhibitory concentration (MIC) of Mono-NHD-Ch and Di-NHD-Ch was perceived at 50 μg/mL against tested microorganisms, except for B. cereus. The MTT test was used to determine the cytotoxicity of the produced materials, which proved their safety to fibroblast cells. The findings suggest that the new N-Alkyl chitosan derivatives might be used as antibacterial alternatives to pure chitosan in wound infection treatments.The objective of this study was to improve the antibacterial activities of chitosan via N-alkyl substitution using 1-bromohexadecane. Mono and di substitution (Mono-NHD-Ch and Di-NHD-Ch) were prepared and characterized using FT-IR, HNMR, TGA, DSC, and SEM. Elemental analysis shows an increase in the C/N ratio from 5.45 for chitosan to 8.63 for Mono-NHD-Ch and 10.46 for Di-NHD-Ch. The antibacterial properties were evaluated against  Chitosan (a deacetylated version of chitin) is one of nature’s polymers commonly derivatized from chitin, and it is second only to cellulose in terms of natural abundance . Chitin It is widely known that chitosan has reasonable antibacterial characteristics against various bacteria, and these properties are linked to the amount of chitosan deposition on bacterium cells ,11. HoweSeveral studies have clearly explained the role of amine groups in antibacterial mechanisms against both Gram-negative and Gram-positive bacteria. Formation of protonated cation derivative simplified the polymers role in distorting the cell wall or penetrating it inside the microorganism cells ,19. FuncThis research focused on the synthesis and characterization of novel chitosan N-Alkyl derivatives. Furthermore, the antibacterial properties of the materials produced were assessed for usage in biomedical applications.Antibiotic resistance in bacteria is prompting scientists to explore new materials to combat the mutations. Therefore, amination, methylation, and other chitosan derivatives were synthesized to improve their antibacterial action ,24,25. TIt is generally recognized that the hydrophilicity of biomaterials has a significant impact on their interactions with living cells. The water absorption values for chitosan and its two alky derivatives, Mono-NHD-Ch and Di-NHD-Ch, are 198.31 ± 9.92, 137.15 ± 6.86, and 104.19 ± 5.21 (%), respectively. The water uptake of the chitosan derivative decreases significantly after coupling with 1-Bromohexadecane as a monosubstituted, whereas it is continuously reduced after di substitution by almost 50%. When amine groups were coupled with 1-Bromohexadecane, hydrophilic sites were substituted with aliphatic long-chain ones, lowering polymer hydrophilicity. The moisture content of chitosan and its alkyl derivatives was tested and calculated as 9.52 ± 0.48 for chitosan, 7.61 ± 0.38 for Mono-NHD-Ch, and 6.18 ± 0.31 for Di-NHD-Ch, indicating their rise in hydrophobic character.Ion exchange capacity was used to estimate the influence of alkyl substitution on chitosan’s surface free amine groups. Pure chitosan records 5.49 ± 0.27 meq/g compared to 5.25 ± 0.26 meq/g for Mono-NHD-Ch and 4.44 ± 0.22 meq/g for Di-NHD-Ch. The measurements focused on the detection of free amine groups on the surface of membranes. The consumption of free amine groups in new derivatization has a direct effect on polymer ion exchange capacity [The derivative of chitosan with alkyl substitution was also identified by elemental analysis, as presented in −1 corresponds to a combination of the stretching vibration of the NH2 and OH groups. A band at 2925 cm−1 refer to the stretching vibration of the C-H bond; the stretching vibration of methylene groups was present at 2850 cm−1. Bands at 1655 cm−1 point out the stretching vibration of the C = O and NH-C = O functional groups. The band at 1459 cm−1 corresponds to -NH2 bending vibration. The peak at 1313 cm−1 is ascribed to C-N stretching vibration. Bands at 1076 cm−1 correspond to C-O-H group stretching.The UV-visible spectra of chitosan and its N-alkyl derivatives are investigated in The TGA of chitosan and its alkyl derivatives was examined, and the results are presented in DSC experiments were used to estimate the thermal characterization, and the results are shown in In S. aureus and B. cereus) (E. coli and P. aeruginosa). Antimicrobial activity inhibition zones were measured, as shown in The antibacterial properties of chitosan and its alkyl derivatives have received a significant amount of interest in recent years. Furthermore, alterations in pathogenic microorganisms that make them resistant to antimicrobial materials have prompted scientists to develop novel antimicrobial materials with potential efficacy against new pathogenic microbes. The antimicrobial properties of chitosan and the two alkyl derivatives were first investigated using an agar-well diffusion technique against Gram-positive and Gram-negative bacteria (3+) and the negative charge of certain bacteria’s cell walls, which causes intracellular components to escape. The second process demonstrates how the molecular weight of chitosan affects its ability to penetrate microorganism nuclei and bind to DNA. As a result, mRNA expression will be suppressed, and protein synthesis will be prevented. The third mechanism is the chelating capacity of chitosan to metal ions such as Ca2+, Mg2+, and Zn2+, which are essential elements for microbial growth and metabolic pathways such as spore production in Gram-positive bacteria. These variables were achieved in the novel medium molecular weight chitosan derivatives. Thus, we propose that the primary methods might be combined to implement the antibacterial potency of the synthesized chitosan derivatives. Previous research said that methyl halide could be used to make a number of alkyl derivatives of chitosan that were more antimicrobial, and the new compounds had good antibacterial, antifungal, antiparasitic, and anticancer properties [The antimicrobial action of chitosan may vary depending on several intrinsic factors, such as the chitosan source; the molecular weight, which influences penetration inside microorganisms; and the synthesis of new chitosan derivatives with novel properties, which usually improve chitosan’s antimicrobial action. Three main chitosan interactions with diverse bacteria have been proposed, differing depending on the cell wall structure and metabolic activity ,36. The operties ,38.Escherichia coli and Staphylococcus aureus. He found that the activity depended on the kind of disaccharide and the degree of substitution [−1 against B. cinerea and P. grisea, respectively [Escherichia coli ATCC 25922 (Gram-negative) and Staphylococcus aureus ATCC 6538 (Gram-positive) bacteria. The study demonstrated that the MDMCMCh showed higher antibacterial activity than TMChC, while MPyMeCh exhibited reduced antibacterial activity against both bacteria [Furthermore, the interaction of the antibacterial polymer with cell wall membranes can be improved by increasing the hydrophobicity of the alkyl side chain of chitosan. This demonstrates the synergistic increase in chitosan activities when mono and di substitutions are used against the microorganisms studied compared to the prime chitosan. They calculated the MIC and bacteria- and fungal-killing abilities of the new chitosan derivatives based on what they learned from this study. Many alkyl derivatives were prepared and showed an improvement in chitosan activity against different bacterial strains. Yang et al. prepared a water-soluble N-alkylated disaccharide chitosan derivative and evaluated it against titution . The Rabectively . Additiobacteria .B. cereus, which required 100 g/mL to halt their growth. Furthermore, pure chitosan did not affect S. aureus at 50 g/mL and required 100 g/mL to do so. Thus, in general, the antibacterial response of the di alkyl substitution is better than that of the mono substitution against all tested microorganisms.The lowest concentrations of chitosan and its two alkyl derivatives (Mono-NHD-Ch and Di-NHD-Ch) that limit the development of the tested strains after overnight incubation were determined as minimum inhibitory concentrations (MICs) in the current study. This method is critical and focused on determining microorganism susceptibilities to our investigated materials and evaluating the antibacterial efficacy of these novel compounds. As shown in E. coli, P. aeruginosa, and S. aureus, respectively, compared to Mono-NHD-inhibition Ch’s percentages of 41.37%, 27.1%, and 24.37% against the same tested microorganisms. At 100 g/mL, both tested derivatives began to inhibit B. cereus, with Mono-NHD-Ch exhibiting a 39.27% inhibition rate and Di-NHD-Ch showing a 43.54%inhibition rate. The inhibition ratios of microbial strains rose as the concentrations of chitosan and its derivatives increased, reaching a maximum at the highest measured concentration (250 g/mL). Accordingly, Di-NHD-Ch at a 250 g/mL dosage inhibited E. coli, S. aureus, and B. cereus almost wholly, with percentages ranging from 97 to 99%. In contrast, the most significant inhibition ratio against P. aeruginosa was 90%. However, using pure chitosan and Mono-NHD-Ch, these complete microbial growth inhibitions were not achieved. This behavior can be explained by the di substation derivative’s increased hydrophobicity, which makes it easier for it to stick to bacterial cell walls and start killing them.Di-NHD-Ch at 50 g/mL showed maximum inhibition ratios of 48.18%, 39.23%, and 24.37% against S. aureus and E. coli, respectively. Against the same bacterial strains, the modified chitosan hydrogel and oxalyl bis 4--yl) benzamide had MICs of 125 and 3.91 g/mL, respectively [In previous research, pure chitosan had 125 and 500 g/mL MIC values against ectively . Anotherectively . It is wectively . Two facE. coli is very resistant to most common antibiotics, so it needs a combination of medicines to control growth and stop biofilms from forming, which can cause cystic fibrosis and even death in people.This research is critical in determining how bacteria react to novel medicinal drugs to minimize doses, intervals, and duration. Several parameters can influence this research, including material concentration, growing circumstances, bacterial density, and time. In this study, 150 g of native chitosan and its two alkyl derivatives, Mono-NHD-Ch and Di-NHD-Ch, were tested for time intervals against the pathogenic organisms previously stated. The findings of the bactericidal experiment are depicted in P. aeruginosa profile shows different behavior according to the tested polymer. Chitosan started to exhibit bacterial inhibition of 55.8% and showed a notable resistance after 4 h. The inhibition rate was 62–67% for the first 3 h, and after that it decreased to 35% at 6 h. Di-NHD-Ch shows the most stable inhibition, which refers to the high sensitivity of P. aeruginosa to Di-NHD-Ch compared to other tested polymers.On the other hand, the S. aureus for the first 3 h, up to 71.2%. The bacterial strain offers resistance after that, showing inhibition activity of around 38% after 6 h. On the other hand, the Gram-positive bacteria B. cereus demonstrates a relatively stable sensitivity that slightly increases in Mono-NHD-Ch.The interaction of the tested polymer shows a significant increase in sensitivity against 1-Bromohexadecane was obtained from Sigma-Aldrich , 1-Methyl-2-pyrrolidinone  was obtained from Alfa Aesar , Chitosan  was obtained from Across Organics , hydrochloric acid (purity 37%) and sodium hydroxide pellets (purity 99–100%) were purchased from Sigma-Aldrich . Sulfuric acid  was purchased from Sigma-Aldrich  and Ethanol  from International Co. for Supp. & Med. Industries . Luria Bertani Broth (LB) was purchased from HIMEDIA LABORATORIES, Mumbai, IndiaWild bacteria were isolated and identified from hospitals in Alexandria, Egypt, according to our published protocol. All collected bacteria showed significant resistance to the multidrug antibiotics ,46.Escherichia coli (KY550380) (E. coli.) and Pseudomonas aeruginosa (KY550378) (P. aeruginosa)) and Gram-positive (Staphylococcus aureus (KY421197) (S. aureus) and Bacillus cereus (B. cereus)) bacteria. All strains were obtained by growing overnight in LB broth medium containing glycerol stocks at 37 °C and 150 rpm .The developing materials’ antibacterial and antifungal performance were tested against Gram-negative (w/v) aqueous NaOH solution, followed by 1-Bromohexadecane, and the solution was stirred for another 6 h at 60 °C. Next, the mono N hexadecanyl chitosan (Mono-NHD-Ch) was precipitated using 200 mL of ethanol, centrifuged, washed twice with acetone on a sintered glass filter, and dried under reduced pressure. The steps were repeated starting from (Mono-NHD-Ch) to prepare the di substitution (Di-NHD-Ch). The products were weighed, and the yield of the reaction was calculated as 62.34% for Mono-NHD-Ch and 51.34% for Di-NHD-Ch according to the following formula:The production of mono and di N-alkyl substituted chitosan followed the procedure with minimal changes , as illuM is the weight of the swollen sample and Mo is the weight of the dry sample, respectively.Water uptake of chitosan and its alkyl derivative samples was determined as a weighed sample (0.06 g) of each tested material and was soaked in distilled water and allowed for equilibrium (up to 24 h). Each sample was then filtered off and carefully bolted with filter paper and with paper tissue that absorbed the excess of adsorbed water on the surface, and each sample was then weighed. Finally, the water swelling was determined by applying the following equation:Mo is the sample’s weight before drying, and M is the sample’s weight after drying.Samples of chitosan and its alkyl derivatives were weighed before and after 3 h of drying at 105 °C. The following formula was used to determine the amount of moisture in the sample:2SO4 solution. The mixture was soaked for 24 h, then filtered, and the aliquot was titrated against a standard solution of sodium hydroxide 0.1 M. Similarly, 20 mL of 0.1 M H2SO4 was tested as a control. The following equation was used to calculate the ionic capacity of chitosan and its alkyl derivatives samples:Ion exchange capacity was measured as a known weight of each test of chitosan or its alkyl derivatives dispersed in (20 mL) of 0.1 M H2SO4 in the absence and presence of chitosan and its alkyl derivatives are 2V and 1V, respectively; a is the normality of NaOH, and w is the sample weight.The volumes of NaOH required for titration of H−1.The functional groups of chitosan and its alkyl derivatives were verified using an FT-IR spectrophotometer . An amount of 1−2 mg of samples were mixed and ground with 200 mg KBr before being scanned in the range of 4000 and 400 cmThe electronic spectrum of the chitosan and chitosan derivatives was estimated using a spectrophotometer (Model Ultrospec 2000). An amount of 0.05 g of chitosan or chitosan derivatives were dissolved in 10 mL of 2% acetic acid solution under heating. The electronic absorbance of the solution in a quartz cell was scanned from 200–600 nm.1H NMR spectra of chitosan and chitosan derivatives were obtained using JEOL 500 MH NMR spectrometer, Japan, at 500.2 MHZ. Polymer solution was prepared by dissolved it in DMSO/TFA and was analyzed by a 5 mm 13C-1H dual probe head at 25 °C. The spectra were accumulated into 32 K data points and processed using exponential multiplication with 2 Hz line broadening into 128 K spectra. For the resulting spectra, 25,000–35,000 scans were accumulated. All spectra were accumulated under identical conditions using power-gated Waltz decoupling with a 25-degree measurement pulse and 1 s pre-pulse delay.NMR spectral analysis: Morphological changes of the sample’s surface were analyzed using a secondary electron detector of SEM (Joel Jsm 6360LA), Japan.Differential scanning calorimetric analysis of chitosan and chitosan derivative samples  was performed in a temperature range of ambient −500 °C at a heating rate of 10 °C/min under nitrogen flow (10 mL/min) using a differential scanning calorimeter device . Morphological changes of the sample’s surface were analyzed using a secondary electron detector of SEM (Joel Jsm 6360LA), Japan.E. coli, P. aeruginosa, S. aureus, and B. cereus, as described by the reported method [The bacterial properties of chitosan and its alkyl derivatives were examined according to agar-well diffusion against d method ,49. For E. coli, P. aeruginosa, S. aureus, and B. cereus was determined using the microtiter plate technique [The effect of various doses of chitosan and chitosan derivatives on the growth of echnique ,51. Firsw/v). After incubation at 37 °C for various durations , the samples were extracted, and 10 µL from each sample was inoculated in 96-well microplates, followed by filling the wells with LB broth up to 200 µL. The microplates were stirred and incubated for 24 h at 37 °C. All experiments were repeated three times, and the turbidities of incubated cultures were measured at 600 nm with a microplate reader, as previously indicated. The average and standard deviations were computed and recorded [A microtiter plate was used to test the bactericidal properties of chitosan and its derivatives. To acquire optical densities (1.8) at 600 nm for all cultures, the overnight cultures of various microorganisms were diluted with an autoclaved LB medium. After that, 1 mL of each cell suspension was combined with 1 mL of chitosan and chitosan derivatives at 1%  was added to each well and incubated for 4 h at 37 °C in a 5% CO2 incubator. The MTT solution  was removed, and the insoluble blue formazan crystals trapped in cells were solubilized in 150 µL of 100% DMSO for 10 min at 37 °C. The absorbance of each well was measured with a microplate reader  at 570 nm to estimate cell viability.A human standard fetal lung cell line (Wi-38) was used to investigate the toxicity of chitosan and its alkyl derivatives (Mono-NHD-Ch and Di-NHD-Ch) according to the method described by Mosmann (1983) . Human WIn this study, two different N-alkyl derivatives of chitosan were prepared and characterized as promising antibacterial polymers by condensing them with 1-Bromohexadecane and obtaining mono and di substitution (Mono-NHD-Ch and Di-NHD-Ch). The obtained results show impressive changes in their properties, which can be summarized as follows:Physicochemical characterization of the new derivatives, such as ion exchange capacity and water uptake, was significantly changed;The N-alkyl derivatives were confirmed by the shift of the polymer characterization peak in their electronic spectrum and the alky signals at the HNMR chart;An FT-IR analysis of the prepared derivatives exhibited a significant new peak;The thermal stability of the chitosan derivatives was enhanced in the new derivatives;The antibacterial evaluation demonstrated that this new N-Alkyl derivative of chitosan exhibited better antibacterial activity than chitosan.


The optimal correction factor was close to 60% of the first-eye prediction error for every formula. A fixed correction factor of 60% of the postoperative refractive error of the first operated eye improves the second-eye refractive outcome better than the methods based on the first eye’s effective lens position back-calculation. A significant interocular biometric dissimilarity precludes the enhancement of the second-eye IOL power calculation according to the first-eye results.The present study is a retrospective, monocentric case series that aims to compare the second-eye IOL power calculation precision using the back-calculated lens position (LP) as a lens position predictor versus using a predetermined correction factor (CF) for thin- and thick-lens IOL calculation formulas. A set of 878 eyes from 439 patients implanted with Finevision IOLs  with both operated eyes was used as a training set to create Haigis-LP and PEARL-LP formulas, using the back-calculated lens position of the contralateral eye as an effective lens position (ELP) predictor. Haigis-CF, Barrett-CF, and PEARL-CF formulas using an optimized correction factor based on the prediction error of the first eye were also designed. A different set of 1500 eyes from 1500 patients operated in the same center was used to compare the basal and enhanced formula performances. The IOL power calculation for the second eye was significantly enhanced by adapting the formulas using the back-calculated ELP of the first eye or by using a correction factor based on the prediction error of the first eye, the latter giving slightly higher precision. A decrease in the mean absolute error of 0.043D was observed between the basal PEARL and the PEARL-CF formula ( In the era where cataract surgery is considered one of the most performed procedures, accurate refractive results are highly awaited . ConsequWe conducted a retrospective data extraction and analysis, including patients who benefited from sequential bilateral cataract surgery from April 2017 to December 2019 by cataract surgeons . All patients bilaterally implanted with Finevision IOLs  were initially included. Exclusion criteria were history of refractive surgery, history of macular disease limiting postoperative visual recovery, previous glaucoma surgery, history of keratoconus or any corneal ectatic disease, presence of central corneal scar. Patients who had at least one exclusion criterion were not included in either set. Included patients were seen preoperatively and at 1 month postoperatively. At each visit, refraction by an optometrist and complete ophthalmological examination by an ophthalmologist were performed. Preoperative biometric measurements were performed using Lenstar 900  and keratometry was calculated using the keratometric index of 1.3375. All cataract extraction surgeries were uncomplicated phacoemulsification with an incision of 2.2 mm.Data were collected in accordance with the tenets of the Declaration of Helsinki, and approval was obtained from the Ethics Review Board of the Canadian Ophthalmic Research Center. The dataset was randomly divided into two samples: both eyes of 439 patients were assigned to a “training set” to develop the formulas based on back-calculated LP and to determine the most effective correction factor (CF) to apply to the formulas based on the latter. A total of 1500 eyes of 1500 patients constituted the “test set” to assess both methods’ performance. In the test set, we randomly included one eye of each patient to avoid measurement bias related to redundancy. Training set data were used to design two modified formulas: PEARL-LP and Haigis-LP. Postoperatively, we determined the “perfect” lens position (LP) for each patient: the value that would lead to the actual postoperative manifest refraction when entered in each IOL calculation formula. Obtained contralateral LP was then used as a lens position predictor, along with classical biometric predictors, in a thin-lens formula (Haigis) and a thick-lens formula (PEARL-DGS). This design led to defining an “a3” coefficient in the Haigis formula and an additional lens position predictor in the PEARL-DGS formula. Intereye AL and anterior curvature radius (ARC) differences were also used as lens position predictors in the latter. We could not design an LP-modified Barret universal II (BU II) formula because the formula is proprietary, thus preventing the back-calculation of the LP. We also used the training set to determine the optimal correction factor (CF) to enhance second-eye refractive prediction using Haigis, BU II, and PEARL-DGS formulas. The respective optimized CF were obtained by analyzing the change in the prediction’s mean absolute error (MAE), between the predicted refraction and the postoperative manifest refraction, with increasing CF (range 0–100%). The optimal CF for each formula was the CF value that resulted in the lowest MAE of prediction for the second eye. For example, if first eye’s implanted IOL power was +20 for a formula prediction of −0.12D and postoperative manifest refraction was −0.75D, resultant PE would be −0.63D and recommended correction would be CF × (−0.63): for a CF of 0.6, recommended correction would be 0.6 × (−0.63) = −0.378D. This value can then be added to the predicted SE of the second eye to determine the corrected predicted SE.p values below the 0.05 threshold, thus indicating a non-normal distribution of mean PE. Statistical comparisons between formulas’ absolute prediction errors were performed using repeated measures analysis of variance . The analysis method proposed by Holladay et al. [p value ≤ 0.05 was considered significant.The prediction error (PE) was defined as the postoperative manifest refraction minus the formula’s prediction: a negative value indicated a myopic error and a positive value a hyperopic one. The mean error (ME) was defined as the mean PE for the entire dataset, and the mean absolute error (MAE) as the mean absolute value of PE for the entire dataset. All calculations and analysis were performed using Python 3.9 with the following libraries: Pandas 1.4.4, Scikit-learn 1.1.2, and SciPy 1.9.1.D’Agostino’s K-squared test performed on formulas’ mean PE consistently yielded y et al.  was not Finally, we analyzed factors that preclude efficient second-eye enhancement. Interocular differences in AL and ARC have been previously reported as limiting factors for second-eye optimization . Even thA total of 1939 patients (3878 paired eyes) were included. A total of 878 eyes from 439 patients were selected to constitute the training set, and 1500 eyes from 1500 patients were discarded from the remaining eyes to avoid using both eyes in the test set, leaving 1500 eyes from 1500 patients. The latter were randomly selected to compare the prediction precision of unmodified base formulas, optimized formulas according to the LP (Haigis-LP and PEARL-LP) and the fixed correction factor CF . The characteristics of the patient’s population are shown in A triple optimization was conducted as described by Haigis: the ELP was retrospectively calculated for each eye of the training set and a linear regression was applied to the ACD and AL to predict this value. The resultant values are one intercept (a0) and two coefficients  . In our p < 0.001) for the LP-adjusted formulas and the CF adjustment in comparison to all three base formulas (the Wilcoxon signed-rank test). The Haigis-CF performed significantly better than the Haigis-LP (p < 0.001) while no statistically significant difference was observed between the PEARL-CF and PEARL-LP. Applying the enhanced Haigis formula using the LP or CF reduced the mean absolute error of the second eye by 0.066D and 0.071D, from 0.302D to 0.236D and 0.231D, respectively. The enhanced PEARL-DGS formula using the LP or CF reduced the MAE of the second eye by 0.040D and 0.043D, from 0.262D to 0.222D and 0.219D, respectively. Finally, optimizing the BU II using a prefixed CF for the second eye reduced the MAE by 0.058D, from 0.284D to 0.226D. The reduction in the MAE with the second-eye method was statistically significant  in 15.47–14.60% of the patients and highly detrimental (decreasing the predictive precision by >0.5D) in 0.53–0.73% of the patients for the PEARL-LP and Haigis-LP, respectively. The CF-modified calculations to the second eye were detrimental in 12.73-14.87-14.40% of the patients and highly detrimental in 0.33-0.80-0.60% of the patients for the PEARL-CF, Haigis-CF, and BU II-CF, respectively . The graThe Haigis formula is a thin-lens formula . It uses the anterior chamber depth (ACD) and AL values as ELP predictors . The PEAFormula-specific adjustments, constant factors, and biometric measurements have been used as optimization methods for the second eye. Li et al. showed that the PEARL-DGS and BUII with formula-specific correction factors improved second-eye refractive outcomes in patients with long AL . RefininAn effective lens position estimation is currently the primary source of the prediction error in cataract surgery . Our goaIn our study, a refractive error higher than 0.50D in the second eye was significantly reduced from 18.2% in the triple-optimized Haigis base formula to 9.6 and 9.47% in the Haigis-LP and Haigis-CF, respectively; from 13.07% in the PEARL-DGS formula to 8.33 and 7.93% in the PEARL-LP and PEARL-CF, respectively; and from 15.74% in the BU II to 9.27% in the BU II-CF. Although it was previously reported that the LP is the leading cause of a prediction error [The second-eye optimization method should not be applied to all cases. Relying on the first-eye refractive outcome to adjust the IOL power selection for the second eye assumes interocular similarity. Our study suggested that if the difference between both eyes was more than 0.5 mm in the AL or more than 0.18 mm in the ARC , employing the PEARL-CF was deleterious. In these cases, unassisted base formulas would result in higher precision in the second eye. Similar results were reported in the literature in eyes with a low interocular correlation , where a partial adjustment in the calculation of the fellow eye would not improve the final outcome . There are some limitations in the present study. First, our study is a monocentric retrospective study, and all the included patients benefited from the implantation of Finevision IOLs . Second, the second-eye refractive refinement is limited by the lens manufacturing tolerance, set by the International Organization for Standardization ISO-11979-2) at ±0.30D for IOLs 15.0D or less, ±0.40D for IOLs more than 15.0D and 25.0D or less, ±0.50D for IOLs more than 25.0D and 30.0D or less, and ±1.0D for IOLs more than 30.0D . Althoug79-2 at ±A fixed correction factor of approximately 60% of the first eye’s prediction error performed best than modifying the IOL formulas by using the back-calculated effective lens position of the first eye as a lens position predictor. According to the first-eye results, the interocular biometric dissimilarity precludes the enhancement of the second-eye IOL power calculation. Further studies are required to assess the benefits of the CF with other types of IOLs, particularly on operated and irregular corneas.


Colorectal cancer (CRC) can be classified into four consensus molecular subtypes (CMS) according to genomic aberrations and gene expression profiles. CMS is expected to be useful in predicting prognosis and selecting chemotherapy regimens. However, there are still no reports on the relationship between the morphology and CMS.This retrospective study included 55 subjects with T2 CRC undergoing surgical resection, of whom 30 had the depressed type and 25 the protruded type. In the classification of the CMS, we first defined cases with deficient mismatch repair as CMS1. And then, CMS2/3 and CMS4 were classified using an online classifier developed by Trinh et al. The staining intensity of CDX2, HTR2B, FRMD6, ZEB1, and KER and the percentage contents of CDX2, FRMD6, and KER are input into the classifier to obtain automatic output classifying the specimen as CMS2/3 or CMS4.According to the results yielded by the online classifier, of the 30 depressed-type cases, 15 (50%) were classified as CMS2/3 and 15 (50%) as CMS4. Of the 25 protruded-type cases, 3 (12%) were classified as CMS1 and 22 (88%) as CMS2/3. All of the T2 CRCs classified as CMS4 were depressed CRCs. More malignant pathological findings such as lymphatic invasion were associated with the depressed rather than protruded T2 CRC cases.Depressed-type T2 CRC had a significant association with CMS4, showing more malignant pathological findings such as lymphatic invasion than the protruded-type, which could explain the reported association between CMS4 CRC and poor prognosis. Colorectal cancer (CRC) is the fourth most diagnosed cancer globally and the second most common in terms of mortality. It is the third most commonly diagnosed malignancy in men and the second in women, with more than 1.8 million new cases and 880,000 deaths reported in 2018 . Thus, cIn 2015, Guinney et al. reported four consensus molecular subtypes (CMS) of CRC according to gene expression . Of thesAmong the morphological types of CRC, depressed-type CRC is more prone to submucosal and lymphatic invasion and has a higher risk of recurrence compared with the other morphological types , 12. Nexhttps://beppu.kyushu-u.ac.jp/geka/wp2/wp-content/uploads/2021/07/disclosure_20210415.pdf and https://www.showa-u.ac.jp/albums/abm.php?d=505&f=abm00031209.pdf&n=210715-1.pdf, respectively). This study was registered in the University Hospital Medical Network Clinical Trials Registry (UMIN000045702). The clinicopathological characteristics of the patients were collected from the electronic medical records of Showa University Northern Yokohama Hospital.Among patients with primary CRC who were surgically resected at Showa University Northern Yokohama Hospital between January 2010 and December 2013, 153 with stage T2 according to the TNM Classification of Malignant Tumors, 8th edition, of the Union for International Cancer Control were enrolled retrospectively. Patients who were diagnosed with Lynch syndrome (n = 3), ulcerative colitis (n = 6), or familial adenomatous polyposis (n = 6) were excluded. Patients lacking detailed clinical data were also excluded (n = 34). We did not include patients who received preoperative chemotherapy or radiotherapy. Tumor morphology was classified according to the Japanese Classification of Colorectal, Appendiceal, and Anal Carcinoma: the 3rd English edition Reviewers' comments:Reviewer's Responses to Questions  Comments to the Author1. Is the manuscript technically sound, and do the data support the conclusions?  The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented. Reviewer #1: NoReviewer #2: Yes**********  2. Has the statistical analysis been performed appropriately and rigorously?   Reviewer #1: YesReviewer #2: Yes**********  3. Have the authors made all data underlying the findings in their manuscript fully available?PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.  The Reviewer #1: YesReviewer #2: Yes**********  4. Is the manuscript presented in an intelligible fashion and written in standard English?  PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.Reviewer #1: YesReviewer #2: Yes**********  5. Review Comments to the Author  Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. Reviewer #1: Mochizuki et al. analyzed 30 depressed type and 25 protruded type T2 colorectal cancers by immunohistochemistry-based CMS classification. CMS4 tumors were exclusive to depressed type and malignant pathological findings were more commonly observed in depressed type tumors compared with protruded type tumors.I do not understand why the authors excluded flat type and ulcerative type lesions from the analysis. The comparison between depressed and protruded types is clearly insufficient to determine the clinicopathological features of depressed type lesions among T2 colorectal cancers. It is possible that CMS4 and malignant pathological findings are significantly less common in protruded type lesions among T2 tumors. Thus, the authors’ conclusion that depressed-type T2 colorectal cancers are significantly associated with CMS4 is not justified by the presented data.Protein symbols should not be italicized.Reviewer #2: Colorectal cancer (CRC) is gradually increasing and major cause of cancer death all over the world. Recent studies have clarified the molecular subtypes of CRC according to gene expression. However, the relationship between molecular profile and morphological types of CRC remains unclear. The authors focused on morphological types, depressed-type T2 CRC and showed significant association with CMS4, which have the worst prognosis among the four CMS subtypes. The findings in this study are very intriguing. However, there were several critical points to be corrected to meet the criteria of this journal.Major Query1. In this study, the authors focused on just depressed and protruded types of the four gross morphological classifications of T2 CRC. Why did the authors select these types? Also, why these 2 types were distinguished by molecular profiling? Please discuss and address about it in Introduction or Discussion section from clinical or molecular point of view.2. The authors showed the relationship between expression of CDX2, KER and malignant grade of CRC. It is likely that both genes are associated with EMT pathway. Please discuss about molecular mechanism in detail. Also, please cite some papers regarding the association between both genes and EMT.3. The authors showed no relationship between tumor location (rectum or not) and each CMS. How about is sidedness of colon cancer? Recently, the impact of sidedness of CRC on tumor immunity has been reported.4. The authors described that depressed CRC has malignant behavior, requiring careful surveillance. How do the authors plan to make use of finding obtained from this study into clinical practice? For example, can depressed-type T2 CRC with CMS4 be candidate factor for the indication for adjuvant chemotherapy? Please describe about clinical perspective of this study.Specific comments1. In introduction section, the authors described about epidemiology of CRC. More worldwide information is needed, not just focusing on Japanese data.2. We can find some typographical errors and different font size (e.g line 227 and 300). Please correct them.3. Who determined the morphological type? Some experts of endoscopist probably did, please specify it in method section because it sometimes depends on endoscopist.4. We miss the data about long-term outcome in this study. The reviewers think it weakness of this study. Please address about it in Discussion section.**********  6. PLOS authors have the option to publish the peer review history of their article (what does this mean?). If published, this will include your full peer review and any attached files.If you choose “no”, your identity will remain anonymous but your review may still be made public.Do you want your identity to be public for this peer review? For information about this choice, including consent withdrawal, please see our Privacy Policy.  Reviewer #1: NoReviewer #2: Nohttps://pacev2.apexcovantage.com/. PACE helps ensure that figures meet PLOS requirements. To use PACE, you must first register as a user. Registration is free. Then, login and navigate to the UPLOAD tab, where you will find detailed instructions on how to use the tool. If you encounter any issues or have any questions when using PACE, please email PLOS at figures@plos.org. Please note that Supporting Information files do not need this step.While revising your submission, please upload your figure files to the Preflight Analysis and Conversion Engine (PACE) digital diagnostic tool,  30 May 2022Dear Editors, We thank the reviewers for their careful assessment of our manuscript and their useful comments. In response to the Reviewers’ comments, we have carefully revised the manuscript point-by-point. We hope that our manuscript is now suitable for publication in the International Journal of PLOS ONE and look forward to hearing from you at your earliest convenience.Yours sincerely, Our responses to the Reviewers’ reports are as follows:----------------------------------------------------------------------Response to the Reviewer----------------------------------------------------------------------Thank you for your careful review of our paper. We have responded to each of your points below.Reviewers' comments:Reviewer #1: Major comments:Mochizuki et al. analyzed 30 depressed types and 25 protruded type T2 colorectal cancers by immunohistochemistry-based CMS classification. CMS4 tumors were exclusive to the depressed type, and malignant pathological findings were more commonly observed in depressed type tumors compared with protruded type tumors.I do not understand why the authors excluded flat type and ulcerative type lesions from the analysis. The comparison between depressed and protruded types is insufficient to determine the clinicopathological features of depressed type lesions among T2 colorectal cancers. CMS4 and malignant pathological findings may be significantly less common in protruded type lesions among T2 tumors. Thus, the authors’ conclusion that depressed-type T2 colorectal cancers are associated considerably with CMS4 is not justified by the presented data. Response: Thank you for your helpful comment. This study excluded flat and ulcerative types and focused our analysis on protruded and depressed types. The adenoma-carcinoma sequence and the de novo pathway, which are the representative carcinogenic pathway of CRC, have been reported to have protruded and depressed morphologies, respectively. To investigate the molecular biological characteristics of these two types, the present analysis focused on the protruded and depressed types.[1]. Attached are figures  on two representative CRC carcinogenesis pathways reported by Kudo et al. As you pointed out, there is a hidden possibility that CMS4 and malignant pathological findings are less common in the protruded type. However, we believe that all CMS4 colorectal cancers were depressed-type colorectal cancers is very impactful data. We want to add flat or ulcerative lesions to our analysis as future work. We have made changes to the Introduction based on your suggestions. We have changed the description of the adoption of morphology as follows:“The adenoma–carcinoma sequence and the de novo pathway, which are representative carcinogenic pathways of CRC, are reportedly associated with the protruded and depressed morphologies, respectively . The present analysis focused on the molecular characteristics of the protruded and depressed types[14].” Minor comments:Protein symbols should not be italicized.Response: Thank you for your comment. We corrected all italicization of proteins.Reviewer #2: Major comments:1. In this study, the authors focused on just depressed and protruded types of the four gross morphological classifications of T2 CRC. Why did the authors select these types? Also, why these 2 types were distinguished by molecular profiling? Please discuss and address about it in Introduction or Discussion section from clinical or molecular point of view.Response: Thank you for your useful comment. In this study, our primary objective was to elucidate the molecular biological characteristics of depressed type colorectal cancer, which is reported to have a high malignant potential, and we referred to the research methods of the pioneering Kudo et al. The adenoma-carcinoma sequence and the de novo pathway, which are representative carcinogenic pathway of CRC, have been reported to have protruded and depressed morphologies, respectively. In order to investigate the molecular biological characteristics of these two types, the present analysis focused on the protruded and depressed types[1]. As you pointed out, there is a hidden possibility that CMS4 and malignant pathological findings are less common in the protruded type. However, we believe that all CMS4 colorectal cancers were depressed-type colorectal cancers is very impactful data. We want to add flat or ulcerative lesions to our analysis as future work. We have made changes to the Introduction based on your suggestions. We have changed the description of the adoption of morphology as follows:“The adenoma–carcinoma sequence and the de novo pathway, which are representative carcinogenic pathways of CRC, are reportedly associated with the protruded and depressed morphologies, respectively . The present analysis focused on the molecular characteristics of the protruded and depressed types [14].” We also believe that molecular profiling has demarcated these two types as follows. First, Kudo et al. reported that depressed CRC is characterized by higher expression of genes related to angiogenesis and EMT. This is consistent with the characteristics of CMS4, and in this study, all of the patients classified as CMS4 were depressed type. Based on these findings, we hypothesized that depressed type CRC is more strongly associated with CMS4 compared to the protruded type. We have added the description of the molecular profiling of morphology as follows:“The concordance of the molecular characteristics of CMS4 with those of depressed CRC, and the classification of all CMS4 cases as the depressed type, demonstrated the strong association between CMS4 and depressed CRC.” 2. The authors showed the relationship between expression of CDX2, KER and malignant grade of CRC. It is likely that both genes are associated with EMT pathway. Please discuss about molecular mechanism in detail. Also, please cite some papers regarding the association between both genes and EMT.Response: Thank you for your very important point. Yu et al. reported that CDX2 inhibits EMT and metastasis of CRC by regulation of Snail expression and β-catenin stabilization through PI3K/Akt/GSK-3β signaling. Kim et al. also reported that keratin loss due to phosphorylation is associated with EMT. Based on the above, we have added the description of CDX2 and EMT as follows:“It has also been reported that CDX2 expression is inversely correlated with the malignancy of CRC [19] and inhibits EMT and metastasis by regulating Snail expression and β-catenin stabilization via PI3K/Akt/GSK-3β signaling [22].” We have added two references reporting on the association between CDX2 and EMT to page 14, line 261, as follows:https://doi.org/10.18632/aging.202537. PMID: 33621200.20. Liu H, Yan R, Liang L, Zhang H, Xiang J, Liu L, et al. The role of CDX2 in renal tubular lesions during diabetic kidney disease. Aging (Albany NY). 2021; 13(5): 6782-6803. https://doi.org/10.3892/mmr.2015.4114. PMID: 26238762.21. Zhang JF, Qu LS, Qian XF, Xia BL, Mao ZB, Chen WC. Nuclear transcription factor CDX2 inhibits gastric cancercell growth and reverses epithelialtomesenchymal transition in vitro and in vivo. Mol Med Rep. 2015; 12(4): 5231-5238. On the other hand, Kim et al. reported that keratin loss due to phosphorylation is associated with EMT. Based on this, we have added the description of KER and EMT and a reference as follows:“On this basis, it has been reported that keratin loss due to phosphorylation is associated with EMT [24].” https://doi.org/10.4062/biomolther.2015.032. PMID: 26157545 24. Kim HJ, Choi WJ, Lee CH. Phosphorylation and Reorganization of Keratin Networks: Implications for Carcinogenesis and Epithelial-Mesenchymal Transition. Biomol Ther (Seoul). 2015; 23(4): 301-312. 3. The authors showed no relationship between each CMS's tumor location (rectum or not). How about is sidedness of colon cancer? Recently, the impact of sidedness of CRC on tumor immunity has been reported.Response: Thank you for your critical comment. We did an additional analysis of the right or left side of the colon and whether it was rectal or not. The results were as follows, with no significant differences. This may be due to the small sample size. Since the relationship between tumor location and immunity is an essential point, we would like to study it again in the future with a more significant number of cases.4. The authors described depressed CRC as malignant behavior requiring careful surveillance. How do the authors plan to use findings obtained from this study in clinical practice? For example, can depressed-type T2 CRC with CMS4 be a candidate factor for the indication for adjuvant chemotherapy? Please describe the clinical perspective of this study.Response: Thank you for your helpful comment. CMS4 has a poor prognosis and is also often at an advanced stage (III-IV)[2]. Considering the high pathologic grade of the depressed type, depressed CRC with CMS4 is likely to require chemotherapy. Based on the results of this study alone, it is not possible to discuss chemotherapy and its effect on chemotherapy due to the small number of cases. In the future, we would like to collect prospective cases and study depressed colorectal cancer, CMS4, and the effects of chemotherapy. We have added the description of a future challenge as follows:“CMS4 is associated with a poor prognosis and is often not detected until the advanced stages (III-IV)[4]. Considering the high pathological grade of the depressed type, depressed CRC with CMS4 usually requires chemotherapy. Although the association between the CMS and long-term outcomes could not be demonstrated due to the small number of cases in this study, we plan to accumulate more cases prospectively as a practical application of this study to determine whether morphology and CMS classification are candidate predictors of the need for adjuvant chemotherapy.” Minor comments:1. In the introduction section, the authors described the epidemiology of CRC. More worldwide information is needed, not just focusing on Japanese data. Response: Thank you for your helpful comment. We have added the description of the epidemiology of CRC as follows:“It is the third most commonly diagnosed malignancy in men and the second in women, with more than 1.8 million new cases and 880,000 deaths reported in 2018 [1].” 2. We can find some typographical errors and different font size (e.g line 227 and 300). Please correct them.Response: Thank you for pointing this out. We have corrected the font.3. Who determined the morphological type? Some experts of endoscopists probably did; please specify it in the method section because it sometimes depends on the endoscopist.Response: Thank you for your helpful comment. We have added the description of the method of determining the morphological type as follows:“The morphological type was retrospectively determined by several endoscopic experts .” 4. We miss the data about long-term outcomes in this study. The reviewers think it is a weakness of this study. Please address it in the Discussion section.Response: Thank you for your helpful comment. Long-term outcomes were analyzed, but the results did not differ due to the small number of cases. Figure B and C of the analysis results are attached. We have added the description of the discussion as follows:“Although the association between the CMS and long-term outcomes could not be demonstrated due to the small number of cases in this study, we plan to accumulate more cases prospectively as a practical application of this study to determine whether morphology and CMS classification are candidate predictors of the need for adjuvant chemotherapy.” https://doi.org/10.1016/j.giec.2008.05.013. PMID: 18674705.1. Kudo SE, Takemura O, Ohtsuka K. Flat and depressed types of early colorectal cancers: from East to West. Gastrointest Endosc Clin N Am. 2008; 18(3): 581-593, xi. https://doi.org/10.1038/nm.3967. PMID: 26457759.2. Guinney J, Dienstmann R, Wang X, de Reynies A, Schlicker A, Soneson C, et al. The consensus molecular subtypes of colorectal cancer. Nat Med. 2015; 21(11): 1350-1356. AttachmentResponse to Reviewers.docxSubmitted filename: Click here for additional data file. 23 Jun 2022
 PONE-D-22-01736R1

 Molecular and clinicopathological features of depressed T2 colorectal cancer

 PLOS ONEDear Dr. Mimori,Thank you for submitting your manuscript to PLOS ONE. After careful consideration, we feel that it has merit but does not fully meet PLOS ONE’s publication criteria as it currently stands. Therefore, we invite you to submit a revised version of the manuscript that addresses the points raised during the review process.Change the title to better reflect the study cohort;Elaborate on the limitation of excluding the flat- and ulcerative-T2 lesions in the Discussion section since these two type of lesions contributed almost equal number as the protruded- and depressed-T2 colorectal cancers of your cohort (49 vs 55).

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Additionally, PLOS ONE offers an option for publishing peer-reviewed Lab Protocol articles, which describe protocols hosted on protocols.io. Read more information on sharing protocols at https://plos.org/protocols?utm_medium=editorial-email&utm_source=authorletters&utm_campaign=protocols.If applicable, we recommend that you deposit your laboratory protocols in protocols.io to enhance the reproducibility of your results. Protocols.io assigns your protocol its own identifier (DOI) so that it can be cited independently in the future. For instructions see: We look forward to receiving your revised manuscript.Kind regards,Peh Yean Cheah, Ph.D.Academic EditorPLOS ONE[Note: HTML markup is below. Please do not edit.]Reviewers' comments:Reviewer's Responses to Questions  Comments to the Author  1. If the authors have adequately addressed your comments raised in a previous round of review and you feel that this manuscript is now acceptable for publication, you may indicate that here to bypass the “Comments to the Author” section, enter your conflict of interest statement in the “Confidential to Editor” section, and submit your "Accept" recommendation.Reviewer #1: (No Response)Reviewer #2: All comments have been addressed**********  2. Is the manuscript technically sound, and do the data support the conclusions?  The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented. Reviewer #1: NoReviewer #2: Yes**********  3. Has the statistical analysis been performed appropriately and rigorously?   Reviewer #1: YesReviewer #2: Yes**********  4. Have the authors made all data underlying the findings in their manuscript fully available?PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.  The Reviewer #1: YesReviewer #2: Yes**********  5. Is the manuscript presented in an intelligible fashion and written in standard English?  PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.Reviewer #1: YesReviewer #2: Yes**********  6. Review Comments to the Author  Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. Reviewer #1: This study analyzes immunohistochemistry-based consensus molecular subtype classification in depressed- and protruded-type T2 colorectal cancers and indicate that depressed-type T2 CRCs are more likely to show the CMS4 phenotype and malignant pathological findings.If the authors want to elucidate the “molecular and clinicopathological features of depressed T2 colorectal cancer”, they should also analyze flat- and ulcerative-type lesions. Otherwise, the authors should extensively revise the manuscript, including the title, to clearly indicate that this study analyzed the differences between depressed- and protruded-type T2 colorectal cancer.The authors added Figure 1, suggesting that protruded and depressed lesions are related to two major tumorigenic pathways. However, what are the tumorigenic processes of flat and ulcerative lesions, which constituted 49 cases in this cohort?Reviewer #2: The authors sufficiently responded to all queries. The reviewer think that this study meets all applicable standards for the ethics of experimentation and research integrity.**********  7. PLOS authors have the option to publish the peer review history of their article (what does this mean?). If published, this will include your full peer review and any attached files.If you choose “no”, your identity will remain anonymous but your review may still be made public.Do you want your identity to be public for this peer review? For information about this choice, including consent withdrawal, please see our Privacy Policy.  Reviewer #1: NoReviewer #2: No**********https://pacev2.apexcovantage.com/. PACE helps ensure that figures meet PLOS requirements. To use PACE, you must first register as a user. Registration is free. Then, login and navigate to the UPLOAD tab, where you will find detailed instructions on how to use the tool. If you encounter any issues or have any questions when using PACE, please email PLOS at figures@plos.org. Please note that Supporting Information files do not need this step.While revising your submission, please upload your figure files to the Preflight Analysis and Conversion Engine (PACE) digital diagnostic tool,  29 Jul 2022----------------------------------------------------------------------Response to the Reviewer----------------------------------------------------------------------Thank you for your careful review of our paper. We have responded to each of your points below.Reviewer's comments:Reviewer #1: This study analyzes immunohistochemistry-based consensus molecular subtype classification in depressed- and protruded-type T2 colorectal cancers and indicate that depressed-type T2 CRCs are more likely to show the CMS4 phenotype and malignant pathological findings.If the authors want to elucidate the “molecular and clinicopathological features of depressed T2 colorectal cancer”, they should also analyze flat- and ulcerative-type lesions. Otherwise, the authors should extensively revise the manuscript, including the title, to clearly indicate that this study analyzed the differences between depressed- and protruded-type T2 colorectal cancer.The authors added Figure 1, suggesting that protruded and depressed lesions are related to two major tumorigenic pathways. However, what are the tumorigenic processes of flat and ulcerative lesions, which constituted 49 cases in this cohort?Response : Thank you for your perceptive comment. We changed the title to “Molecular and clinicopathological differences between depressed and protruded T2 colorectal cancer.” As you have concerned in the original manuscript, we have excluded findings related to the flat and ulcerated types, which should be discussed as the major limitation in this paper. Therefore, we have added a paragraph to discuss it as follows:“Fourth, we excluded flat type and ulcerative type cases of CRC. In this study, we focused on the two hypotheses of carcinogenic pathways in CRC: protruded type by the adenoma-carcinoma sequence and depressed type by the de novo pathway. We speculate that lesions with endoscopically depressed localization rarely have an adenoma component in the pathology[11] and take their carcinogenic pathway. The flat type and ulcerative type are comprised of a mixture of both protruded and depressing elements and a mixture of tumors that have followed many carcinogenic pathways, such as those originating from serrated lesions[30] or granular-type laterally spreading tumors (G-LST)[31]. Therefore, it was appropriate first to clarify the two representative carcinogenic pathways of CRC, the adenoma-carcinoma sequence and the de novo pathway, and clarification of the flat and ulcerative type was considered an issue to be addressed later. We excluded flat and ulcerated type T2 CRCs. However, there were 49 cases of flat and ulcerated type, which is not negligible and may have a large selection bias. Serrated pathway carcinogenesis has been reported to include MSI carcinomas[30], and G-LST has been reported to have a High prevalence of CpG island methylator phenotype-high[31]. These are consistent with the characteristics of CMS1. Perhaps the morphology excluded in this study includes CMS1 cases, which may have represented only a minority of cases in this study.” https://doi.org/10.14309/ctg.0000000000000269. PMID: 33512809.11. Kudo SE, Kouyama Y, Ogawa Y, Ichimasa K, Hamada T, Kato K, et al. Depressed Colorectal Cancer: A New Paradigm in Early Colorectal Cancer. Clin Transl Gastroenterol. 2020; 11(12): e00269. https://doi.org/10.1111/his.12564. PMID: 25263173.30. O'Brien MJ, Zhao Q, Yang S. Colorectal serrated pathway cancers and precursors. Histopathology. 2015; 66(1): 49-65. https://doi.org/10.1053/j.gastro.2006.04.027. PMID: 16890591.31. Hiraoka S, Kato J, Tatsukawa M, Harada K, Fujita H, Morikawa T, et al. Laterally spreading type of colorectal adenoma exhibits a unique methylation phenotype and K-ras mutations. Gastroenterology. 2006; 131(2): 379-389. AttachmentResponse to Reviewers.docxSubmitted filename: Click here for additional data file. 11 Aug 2022Molecular and clinicopathological differences between depressed and protruded T2 colorectal cancerPONE-D-22-01736R2Dear Dr. Mimori,We’re pleased to inform you that your manuscript has been judged scientifically suitable for publication and will be formally accepted for publication once it meets all outstanding technical requirements.Within one week, you’ll receive an e-mail detailing the required amendments. When these have been addressed, you’ll receive a formal acceptance letter and your manuscript will be scheduled for publication.http://www.editorialmanager.com/pone/, click the 'Update My Information' link at the top of the page, and double check that your user information is up-to-date. If you have any billing related questions, please contact our Author Billing department directly at authorbilling@plos.org.An invoice for payment will follow shortly after the formal acceptance. 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For more information, please contact Kind regards,Peh Yean Cheah, Ph.D.Academic EditorPLOS ONEAdditional Editor Comments :rather than protruded T2 CRC cases.Please change this sentence in Abstract (line 43) to: More malignant pathological findings such as lymphatic invasion were associated with the depressed different carcinogenic pathway.Please change this sentence in Discussion (line 323) to: We speculate that lesions with endoscopically depressed localization rarely have an adenoma component in the pathology[11] and take Reviewers' comments: 2 Sep 2022PONE-D-22-01736R2 Molecular and clinicopathological differences between depressed and protruded T2 colorectal cancer Dear Dr. Mimori:I'm pleased to inform you that your manuscript has been deemed suitable for publication in PLOS ONE. Congratulations! Your manuscript is now with our production department. onepress@plos.org.If your institution or institutions have a press office, please let them know about your upcoming paper now to help maximize its impact. If they'll be preparing press materials, please inform our press team within the next 48 hours. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information please contact plosone@plos.org. If we can help with anything else, please email us at Thank you for submitting your work to PLOS ONE and supporting open access. Kind regards, PLOS ONE Editorial Office Staffon behalf ofDr. Peh Yean Cheah Academic EditorPLOS ONE


As a member of interleukin-12 family, interleukin-35 (IL-35) plays an important regulatory role in immune response. The relationship between IL-35 and idiopathic membranous nephropathy (IMN) is still unclear, and the purpose of this study is to clarify the relationship between IL-35 and disease activity and remission of IMN.This study was a single-center, retrospective study in which all patients were diagnosed with IMN by renal biopsy or aPLA2R titer and treated with Mahuang Fuzi and Shenzhuo Decoction (MFSD). A follow-up was conducted with the endpoint of clinical complete or partial remission (CR+PR). Levels of serum IL-35 were measured and its relationship with IMN remission were analyzed. The regulatory T cell (Treg) and inducible IL-35 producing Tregs (iTR35) in peripheral blood of IMN patients were detected by flow cytometry.vs 83.7%, P<0.01). The baseline IL-35 level in remission group was higher than that in NR group . Cox regression analysis showed that baseline IL-35 level was a independent risk factor for IMN remission . Patients with baseline IL-35 lower than the lower quartile (≤145.49 pg/ml) had an average remission time twice as long as those with baseline IL-35 higher than the upper quartile (> 203.05 pg/ml) . The baseline IL-35 can predict the remission time of IMN patients with either aPLA2R positive (AUC=0.673) or negative (AUC=0.745). Analysis of 18 patients with IMN showed that IL-35 level had a higher correlation with iTR35, but not Treg .A total of 76 IMN patients (age 51.95 ± 13.29) were followed-up for 18  months. The level of serum IL-35 in all patients increased after treatment, but the degree of increase in remission group was significantly higher than that in no remission (NR) group (117.6% The level of IL-35 in patients with IMN showed an increasing trend with the progress of treatment, and the baseline IL-35 could predict the remission time of IMN patients, including those patients with negative aPLA2R. The level of IL-35 is related to the number of iTR35 cells. Membranous nephropathy is a kind of immune-mediated glomerular disease, which is characterized by immune deposits in glomerular basement membrane and its diffuse thickening. It is one of the main pathological types of nephrotic syndrome . ClinicaAt present, PLA2R is the main autoantigen of IMN patients, accounting for about 70% . There iInterleukin-35 (IL-35), as a member of IL-12 family, is a heterodimer composed of two chains, EBI3 and p35 . AlthougRecent studies have shown that the level of serum IL-35 is related to autoimmune diseases. In rheumatoid arthritis (RA), serum IL-35 was negatively correlated with disease activity, and the level of IL-35 increased after remission. The level of IL-35 also increased in RA patients who received immunosuppressive therapy . The aniThere are few studies on IL-35 and IMN. Roccatello et al.  reportedThis study is a retrospective cohort study, including 76 IMN patients who were treated in Beijing Hospital of Traditional Chinese Medicine from Oct, 2016 to Oct, 2021. The inclusion and exclusion criteria were as follows: (1) Patients with MN diagnosis by renal biopsy and/or positive aPLA2R with nephrotic syndrome were included. (2) Exclude MN patients secondary to infectious or other systemic autoimmune diseases, or complicated with serious diseases or dysfunction of other organs . ProteinThis study was approved by the Ethics Committee of Beijing Hospital of Traditional Chinese Medicine affiliated to Capital Medical University , and all the participants signed the informed consent form.A total of 13ml venous blood of each IMN patient were collected. Of these, 3ml was used for the detection of serum IL-35, and the other 10ml were placed in heparinized test tubes for isolation of mononuclear cells. Peripheral blood mononuclear cells (PBMCs) were isolated from heparinized peripheral blood by Lymphoprep™  density gradient centrifugation, following the standard protocol.Serum IL-35 were assayed using a human IL-35 ELISA kit  according to user manual. The IL-35 ELISA kit has a dynamic range of 15.63-1000 pg/mL, and a sensitivity of 1 pg/mL. Detection of serum aPLA2R titer was performed using ELISA kit  according to the manufacturer’s recommendation, a value ≥14 RU/ml was considered as positive. ELISA measurement of all samples was carried out twice and then calculate average value.+CD4+CD25+Foxp3+T cells. iTR35 cells were defined as CD3+CD4+Foxp3-EBI3+IL-12p35+T cells. For the intracellular cytokine analysis, PBMCs were stimulated with Leukocyte Activation Cocktail  at 37°C for 6 hours prior to staining. Cells were surface stained with anti-CD3-PE-Cy7, anti-CD4-FITC, anti-CD25-BV421  at 4°C for 30 min. And then further fixed and permeabilized . Subsequently, the cells were intracellular stained with anti-Foxp3-APC, anti-EBI3-PE, anti-IL-12p35-Percp  at 4°C for 30 min. Before detection, the living and dead cells were identified by Zombie NIR Fixable Viability Kit . All samples were processed by FACSCanto™ II .The number of Treg and iTR35 cells were performed by Flow cytometry. Treg cells were defined as CD3Continuous variables that conform to normal distribution are described by mean standard deviation, while those that do not conform to normal distribution are described by median and interquartile spacing. T-test of independent sample is used to compare the difference between two groups of normal distribution, and Mann-Whitney U test is used to compare the difference between two groups of non-normal distribution. The classified variables were expressed by ratio, and the comparison between the two groups was made by chi-square test. The correlation analysis adopts Pearson  correlation analysis and Spearman  rank correlation analysis.p value. The Cox regression analysis establishes the prediction model of progress to the outcome.The ROC analysis was used to compare the area under the curve between serum IL-35 and aPLA2R titer, to evaluate its predictive ability of clinical response  as an immune response index. Remission status analysis was performed using Kaplan–Meier method. Log-rank test was used to statistically compare the curves and P < 0.05 was considered statistically significant.For statistical analysis the SPSS statistical software package was used . The graphs were performed by GraphPad Prism 9 . Some data visualization and statistical analysis were done using R, including the ggplot2 package. P<0.05). In addition, serum IL-35 levels of 22 healthy controls were also detected, and it was found that IL-35 levels of IMN patients were significantly higher (P<0.05) than those of healthy controls , while the ascensional range in IL-35 were different between the two groups. It was higher in remission group (117.6%) than that in no remission group (83.7%) (P<0.05). This suggested that the IL-35 level of the same IMN patient shows an increasing trend during the treatment, and the increased degree of IL-35 in the remission group is higher than that in the no remission group.According to whether they are in remission at the end of follow-up, IMN patients were divided into two groups: remission group (CR+PR) (n=59) and no remission group (NR)(n=17). Serum IL-35 levels were measured before and after treatment. Our results showed that serum IL-35 levels were 147.39 (107.38-187.82) pg/mL and 245.76 (122.15-361.76) pg/mL before and after treatment in the NR group, respectively. The CR+PR group was 175.86 (150.20-216.51) pg/ml and 382.65 (162.87-449.94) pg/ml, respectively (P<0.05). While after 6 months, the level of IL-35 in CR+PR group gradually decreased, and the median value was like that in NR group. In NR group, the change of IL-35 level was smaller than that in CR+PR group. IL-35 level increased slightly at 0-6 months, and then gradually decreased. The levels of IL-35 in the two groups had no difference after the 12th and 24th or longer months  than that in NR group (P<0.01) and ALB was higher (P<0.01) in remission group. But there was no significant difference in other indexes  , which means that for every 10 pg/ml increase in baseline serum IL-35 level, the remission rate of IMN patients increases by 8.1% , while Group4 was significantly different from Group1  : Group1 (≤145.49pg/ml), Group2 (145.49-173.54pg/ml), Group3 (173.54-203.05pg/ml), and Group4 (>203.05pg/ml). There was no significant difference between Group2, Group3 and Group1  and > 203.05 pg/ml (Group 4). Kaplan-Meier survival analysis was used to compare the remission time between the two groups. The results showed a median remission time of 12 months in IMN patients with Group 1(n=18) and 24 months with Group 4 (n=19), and the remission time of group 4 is twice if that of group1  values of IL-35 was 0.673, the aPLA2R titer was 0.521, and combined diagnosis was 0.686, suggesting that a combination of IL-35 and aPLA2R titer was more effective than IL-35 and aPLA2R titer alone  and iTR35 (CD3+CD4+Foxp3-EBI3+IL-12p35+T cells) in PBMCs from 18 IMN patients, and the detected flow analysis image was shown in P>0.05). However, there was a positive correlation between IL-35 and iTR35 cells   and remission , and negatively correlated with 24hUTP , nephrotic syndrome state  and remission time . the detail is shown in Moreover, we also analyzed the correlation between IL-35 and other parameters. And we found that serum IL-35 levels were positively correlated with ALB  (P<0.05). There was no obvious change in renal function in most patients before and after treatment, and no serious adverse reactions occurred in all patients during treatment  follow-up, the remission rate (CR+PR) of IMN patients treated with the MFSD regimen reached 61.4% . Among tP=0.254) . During reatment . Thus, treatment . A randoOur research shows that the level of IL-35 in patients with IMN in remission stage is significantly higher than that in active disease, which may be related to the shift from immune state to immunosuppression. It is worth noting that the level of IL-35 in IMN patients is always higher than that in healthy controls, whether in remission or active period, suggesting that as an autoimmune disease, IMN patients’ autoimmune response remains active, even if the disease is relieved. Mohd shukri et al.  also fouin vivo, that is, from hyperimmunity to immunosuppression, then it can be expected that the remission of IMN patients will be achieved accordingly. The immune status of patients who have not been relieved has been relieved to some extent, but it still takes some time to achieve clinical remission. Roccatello et al.  rat model, but its mechanism is still under study . CombineIn conclusion, our research has confirmed the change of IL-35 in IMN patients. The baseline IL-35 level reflects the immune status of patients, and patients with higher baseline IL-35 can be relieved more quickly. We also show that the therapeutic target of the MFSD regimen may be different from the traditional immunosuppressive regimen, which provides more choices for the treatment of IMN.The following shortcomings exist in this study: this study is a retrospective study, and a larger number of patients would have been more beneficial in drawing the conclusions of this study. In addition, the number of samples for detecting Treg and iTR35 in PBMC of patients with IMN is a little less, due to the difficulty in PBMC preservation.The original contributions presented in the study are included in the article/This study was reviewed and approved by Medical Ethics Committee of Beijing Hospital of Traditional Chinese Medicine. The patients/participants provided their written informed consent to participate in this study.BL and HR were responsible for conception of the study. HD, YG, XD, HJ, and YH are responsible for data analysis and interpretation. WL, ZF, ZD, QZ and GH were responsible for conception of the study and responsible for critical revision of important content, and undertook part of the data analysis and interpretation work. NZ was involved in drafting the manuscript. BL and HR were responsible for approving the final version to be published and agree to be responsible for all aspects of the work and to ensure that issues relating to the accuracy or completeness of any part of the work are properly investigated and resolved. The authenticity of the original data in this paper has been confirmed by BL and HD, and they are responsible for this. All authors contributed to the article and approved the submitted version.This work was supported by grants from the National Key Research and Development Project of China (no. 2019YFC1709402 to BL), National Natural Science Foundation of China , and Capital’s Funds for Health Improvement and Research (No. 2020-2-2234 to BL).The authors sincerely acknowledge the support of Dr. Huihui Liu, Xin Liu for their support of flow cytometry for evaluation of immunity during this work.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


Background: Maternal smoking during pregnancy has adverse health effects on the offspring, including lower birth weight and increased risk for obesity. These outcomes are also influenced by common genetic polymorphisms. We aimed to investigate the combined effect of maternal smoking during pregnancy and genetic predisposition on birth weight and body mass index (BMI)-related traits in 1,086 children of the Human Early Life Exposome (HELIX) project.Methods: Maternal smoking during pregnancy was self-reported. Phenotypic traits were assessed at birth or at the age of 8 years. Ten polygenic risk scores (PRSs) per trait were calculated using the PRSice v2 program. For birth weight, we estimated two sets of PRSs based on two different base GWAS summary statistics: PRS-EGG, which includes HELIX children, and PRS-PanUK, which is completely independent. The best PRS per trait (highest R2) was selected for downstream analyses, and it was treated in continuous or categorized into three groups. Multivariate linear regression models were applied to evaluate the association of the explanatory variables with the traits of interest. The combined effect was evaluated by including an interaction term in the regression models and then running models stratified by the PRS group.Results: BMI-related traits were correlated among them but not with birth weight. A similar pattern was observed for their PRSs. On average, the PRSs explained ∼4% of the phenotypic variation, with higher PRS values related to higher trait values . Sustained maternal smoking was associated with lower birth weight and higher BMI and related traits . We identified a gene by environment (GxE) interaction for birth weight between sustained maternal smoking and the PRS-EGG in three groups , which was not replicated with the PRS-PanUK . Finally, we did not find any statistically significant GxE interaction for BMI-related traits .Conclusion: Sustained maternal smoking and the PRSs were independently associated with birth weight and childhood BMI-related traits. There was low evidence of GxE interactions. Maternal tobacco smoking during pregnancy remains a great concern in public health, with 4.2–18.9% of European mothers smoking during the pregnancy period . ChildreAnother application of PRSs is the investigation of pleiotropy. Pleiotropy is defined as the shared influence of a genetic variant on more than one unrelated phenotype and is common in complex traits . For insFinally, PRS can also be used to stratify the environmental risk to disease by conducting gene by environmental (GxE) interaction analyses . Some stIn this study, we aimed to investigate the combined effect of maternal smoking during pregnancy and genetic predisposition on BW and BMI-related traits. We calculated several PRSs for these traits in children of the Human Early Life Exposome (HELIX) project and then tested their GxE interactions with maternal smoking during pregnancy.https://www.projecthelix.eu/) is a collaborative project across six established and ongoing longitudinal population-based birth cohort studies in six European countries: EDEN - Étude des Déterminants pré et postnatals du développement et de la santé de l’Enfant (France); Rhea - The Rhea Mother–Child Study in Crete (Greece); KANC - Kaunas Cohort (Lithuania), MoBa - Norwegian Mother, Father and Child Cohort Study (Norway), INMA - INfancia y Medio Ambiente (Spain), and BiB - Born in Bradford (United Kingdom) (HELIX (Kingdom) . In summ.PRS were calculated in the 1,155 European ancestry children from the HELIX project with genetic data, and the GxE interaction analyses were performed in 1,086 children with genetic, phenotypic, and exposure data .Maternal tobacco smoking was self-reported by the mother during different trimesters of pregnancy, from which we created two variables: any and sustained maternal smoking. Any maternal smoking was categorized as “smokers” (mothers who smoked at any point during pregnancy) and “non-smokers” . Sustained maternal smoking was categorized as “non-smokers,” “non-sustained smokers” (mothers who smoked only during the first trimester), and “sustained smokers” (mothers who smoked until the third trimester).Exposure to second-hand smoke in childhood was assessed through a harmonized questionnaire administered to the parents at the age of ∼8 years. Children were classified as “exposed” if they were exposed to second-hand smoke at home or in other indoor spaces and as “unexposed” if they were not exposed at all.BW (in grams) was collected as part of the study protocol of each cohort and harmonized in the context of the European Study of Cohort for Air Pollution Effects (ESCAPE) project . Wheneve2) was calculated and converted into age- and sex-standardized z-scores (zBMI) using the international World Health Organization (WHO) reference curves . Genotype calling was performed using the GenTrain2.0 algorithm based on a custom cluster file implemented in the GenomeStudio software and annotation with the GSAMD-24v1-0_20,011,747_A4 manifest.Child peripheral blood DNA samples were collected at the age of ∼8 years. Genome-wide genotyping was performed using the Infinium Global Screening Array (GSA) MD version 1 (Illumina) at the Human Genomics Facility, Erasmus MC .Sample quality control was performed using the PLINK program . The folVCF format, and variants were aligned with the reference genome. The phasing of the haplotypes was performed with Eagle v2.4 and the imputation with minimac4. Chromosome X was imputed, including PAR and non-PAR regions. In total, we retrieved 40,405,505 variants after imputation. Several quality control filters were applied to the imputed dataset: 1) imputation accuracy (R2) <0.9, 2) MAF <1% and 3) HWE p-value <1E-06, giving rise to a final post-imputation dataset consisting of 1,304 samples and 6,143,757 variants (human genome build GRCh37/hg19 and plus strand). The first 20 principal components were computed from the GWAS data of European ancestry children using PLINK with the LD clumping option.Genome-wide genotype imputation was performed with the Imputation Michigan server using the Haplotype Reference Consortium (HRC) cosmopolitan panel, version r1.1 2016. Before imputation, PLINK GWAS data were converted into . PRSs were calculated operating with the average score method, which computes the score as a sum of the summary statistics for the effective allele multiplied by the number of effective alleles observed, divided by the number of alleles included in the PRS. SNP clumping was set at r2 > 0.1, and the rest of the arguments were left as default in PRSice v2. For each trait, we calculated 10 PRSs based on ten different p-value thresholds (Pt) of the base GWAS for SNP inclusion: Pt = {0.00000005, 0.000005, 0.0001, 0.001, 0.01, 0.05, 0.1, 0.2, 0.5, and 1}. For BW, we computed two sets of 10 PRSs: one using the EGG base GWAS (which includes around 25% of the HELIX children) (PRS-EGG) and the other using the PanUK Biobank base GWAS  (PRS-PanUK).PRSs for BW, BMI, WC, and FM were computed for 1,155 children of the European ancestry using the PRSice v2 program and the imputed and quality controlled genetic data . Summarip-value thresholds (Pt) was selected using PRSice v2. In particular, linear regression models were computed between the phenotypic trait and each PRS adjusting for the 10 first GWAS principal components (PCs). The PRS with the highest model-fit (R2) was considered the best and used in downstream analyses. To control for overfitting due to parameter optimization , the association between the best PRS and the phenotypic trait was corrected by performing 10,000 permutations of the phenotype and calculating an empirical p-value.The optimal PRS for each trait among the 10 PRSs calculated with different Finally, the best PRSs were centered and scaled by subtracting the mean and divided by the SD. They were analyzed as continuous variables and categorized into three groups: PRS-low (<25% percentile), PRS-mid (from 25 to 75% percentile), and PRS-high (>75% percentile). PRS was constructed based on the weights of the base GWAS; thus, the PRS group representing the highest genetic risk for zBW is the PRS-low, while for the other traits is the PRS-high group.Frequencies  and means and SDs (for continuous variables) were calculated. The cross-correlation of the PRSs and phenotypic traits was calculated with Pearson’s correlation coefficients and represented in heatmaps with the corrplot R package .Phenotypic trait = PRS-continuous + any maternal smoking during pregnancy + covariatesPhenotypic trait = PRS-continuous + sustained maternal smoking during pregnancy + covariatesPhenotypic trait = PRS-groups + any maternal smoking during pregnancy + covariatesPhenotypic trait = PRS-groups + sustained maternal smoking during pregnancy + covariatesTo test the association of the PRS and maternal smoking during pregnancy with phenotypic traits in the selected 1,086 children of the study, we fitted several linear regression models adjusted for covariates:Covariates for zBW were sex, gestational age, 10 first GWAS PCs, and maternal education. Covariates for zBMI-related traits were 10 first GWAS PCs, maternal education (in three levels), and second-hand smoke in childhood. Maternal education, which is associated with socioeconomic status and maternal age, was included as a covariate as it is a potential confounder for maternal smoking during pregnancy.Phenotypic trait = PRS-continuous + any maternal smoking during pregnancy + PRS-continuous * any maternal smoking during pregnancy + covariatesPhenotypic trait = PRS-continuous + sustained maternal smoking during pregnancy + PRS-continuous * sustained maternal smoking during pregnancy + covariatesPhenotypic trait = PRS-groups + any maternal smoking during pregnancy + PRS-groups * any maternal smoking during pregnancy + covariatesPhenotypic trait = PRS-groups + sustained maternal smoking during pregnancy + PRS-groups * sustained maternal smoking during pregnancy + covariatesEffect sizes are expressed as the change in the z-score of the phenotypic trait (where each unit represents one standard deviation from the mean considering age and sex), by maternal smoking status (non-smokers as reference), by PRS group (PRS-low as reference), or by one standard deviation of the PRS (when treated in continuous). The effect modification of the PRSs on the association between maternal smoking and the phenotypes was tested by including an interaction term between these variables in the linear regression model:p-value). For statistically significant GxE interactions, models stratified by the PRS group were run.Partial F-tests were used to determine statistically significant differences between models with and without the interaction term  and of excluding non-preterm children. We also tested the effect on zBMI-related traits by not adjusting the models for childhood second-hand smoke and by excluding children unexposed to second-hand smoke.https://github.com/georginafp/analysis_GxE).All the analyses were conducted in R (version 4.0.3), and scripts can be found in a GitHub repository  or own distributions (zWC and zFM). In total, 16.9% of the mothers reported having smoked at some point during pregnancy, and 9.7% were classified as sustained smokers as they smoked during the whole pregnancy period. Childhood exposure to second-hand smoke at home or other places was 36.8%. The correlation between pregnancy and childhood exposure was ∼0.4 and has been described elsewhere in detail (The description of the main variables of the HELIX children is shown in n detail . The desR2), and the associations of the best PRS in quantiles are shown in p-values for the association of the best PRSs with phenotypic traits were 9.99E-05. The cross-correlation of the PRSs and phenotypic traits is shown in Ten PRSs were calculated per phenotypic trait for the 1,155 HELIX children. The distribution of the PRSs, the total phenotypic variation explained  . The associations were only observed among children born from sustained smokers  and not among children born of non-sustained smokers . In particular, children exposed to sustained smoking during pregnancy had 0.500  lower zBW (adjusted for the PRS-PanUK), 0.321 (0.125) higher zBMI, 0.215 (0.099) higher zFM, and 0.280 (0.010) higher zWC than unexposed children. The effects of sustained maternal smoking on the phenotypic traits in the models including the PRS in three groups were similar .In the model including the continuous PRS, any maternal smoking in pregnancy was at least marginally associated with lower child zBW and higher zBMI, zWC, and zFM (64E-02) . The assimilar .p-values <5.55E-08)  were positively associated with the respective phenotypic traits (.55E-08) . The samp-values interaction = 0.052 and 0.01, for any and sustained smoking, respectively)  and -0.655 (0.157) for children in the PRS-low and in the PRS-mid groups, respectively). In contrast, the effect of sustained smoking was negligible in children within the PRS-high group (-0.097 (0.195))    . Materna(0.195)) . Howeverively) ,. Finally >0.237) .R2 values explained by the PRS-EGG in the subsets of children without overlap were 4.8% (without INMA) and 4.2% (without INMA and MoBa). Second, we repeated the analyses of sustained maternal smoking in relation to zBW without preterm children, and the results did not change substantially  of the base GWAS using PRSice v2, which is one of the most widely used tools to compute PRSs. The best PRSs were robustly associated with the phenotypic traits but only explained ∼4% of the phenotypic variation (R2). In HELIX, the variation explained by the PRS of BMI that included 60,993 SNPs (R2 = 4.7%) was in the range or slightly lower than previous estimations in children , in ado 97 SNPs ; R2 = 5376 SNPs ; R2 = 6.2 M SNPs ; R2 = 71 M SNPs ). In Khe1 M SNPs . In our Regarding BW, previous studies have reported that a PRS for BW including 62 SNPs from the EGG consortium explains between 2 and 4.9% of the phenotypic variation . In HELI2 more in childhood than children in the PRS-low. Offspring born from sustained smoker mothers had a mean reduction of BW of ∼173 g and a mean increase of BMI of ∼0.742 kg/m2, compared to offspring of non-smokers. Of note, the comparison of effect sizes between the PRS and maternal smoking is subjected to the PRS categorizations we applied  and to measurement error, which is likely larger for maternal smoking .For all the traits, having a PRS above the 75th percentile (PRS-high) compared to having a PRS below the 25th percentile (PRS-low) had a stronger effect (in absolute terms) than the exposure to tobacco smoke during the whole pregnancy. Children with a PRS-high level weighed ∼266 g more at birth and had ∼1.573 kg/mThe GxE analyses showed low evidence of interactive effects for BMI-related traits between the PRSs and maternal smoking during pregnancy. In contrast, other studies have reported GxE interactions between BMI genetic predisposition with a PRS including 2.1 M SNPs, and geographic region in Europe, parental education, fiber intake, or screen time , and betWe detected an interaction for BW between sustained maternal smoking and the PRS-EGG, which was not confirmed when using the PRS-PanUK. Compared to others, children with a genetic predisposition to high BW (EGG PRS-high group) seemed to be protected against the adverse effects of sustained maternal smoking on BW. Surprisingly, the adverse effect of maternal smoking on BW was stronger in children in the PRS-mid group than in children in the PRS-low group, likely due to the low numbers in each group. The interaction did not reach statistical significance when treating the PRS in continuous. Moreover, despite the correlation between the two PRS for BW (r = 0.48), the protective effect of the genetic background was not observed in children in the PRS-high group computed with the PanUK base GWAS. The EGG base GWAS data includes around 25% of the HELIX children, which represent <0.2% of the base sample . This isThe results should be considered in light of some limitations. The first one is that PRS predictability depends on the quality of the reference GWAS . Second, due to the lack of reference GWAS in non-European populations, we only calculated the PRSs in European ancestry HELIX children. Third, HELIX is a pediatric population composed of children from 5 to 12 years, while the base GWAS for BMI-related traits were conducted in adults. Hence, genetic variants having specific effects only in adults might have biased our findings, while variants having specific effects only in children, and thus not considered in the PRS, might have decreased the PRS predictability. In any case, our PRSs could predict part of the variation of the traits, indicating that some genetic factors seem to be stable over life, as described before for BMI . Fourth,Nonetheless, our study also has some strengths. First, HELIX is a well-characterized cohort in which phenotypic measurements were obtained under extremely harmonized protocols. Second, we have focused on maternal smoking during pregnancy, a well-characterized exposure for which strong effects on offspring health outcomes have been described. Finally, this study could serve as a basis for larger studies combining data of different cohorts that aim to investigate GxE interactions.In summary, maternal smoking during the whole pregnancy period, but not only at the beginning, was related to lower BW and higher BMI-related traits in childhood. The PRSs were associated with the phenotypic traits, but they explained a low proportion of the variation. There was low evidence for GxE interactions, except for BW, where children in the highest PRS group seemed to be protected against the damaging effects of sustained maternal smoking. However, this was not validated when using another PRS for BW, and thus, it requires further investigation.